Article Figures & Data
Figures
- Figure 1.
MAPINS identifies flanking genomic DNA sequences around insertion sites. Paired-end 101-bp reads are composed of four different groups. They are reads completely aligned to the Chlamydomonas genome (A, black thin lines); reads completely aligned to the cassette sequence (B, orange thin lines); and reads that are chimeric between Chlamydomonas genomic sequences and the cassette sequence (C and D, mixed black-orange lines). All reads in C have the Chlamydomonas sequence at the 5′ end, and all reads in D have the cassette sequence at the 5′ end. Reads #1 to #6 represent different patterns of chimeric DNA from the 5′ to 3′ end. Step 1, All reads are first aligned to the Chlamydomonas reference genome (black thick line), and all completely aligned reads (group A) are discarded (red dashed box). Two parallel analyses are performed for the remaining reads (groups B, C, and D; red solid box) in steps 2 and 3. Step 2, They are aligned to the cassette sequence (orange thick lines), and all completely aligned reads (group B) are discarded (magenta dashed box). Reads in groups C and D are retained (magenta solid shaded box). Step 3, They are truncated from the 3′ end to 45 bp long in length. These 45-bp reads are aligned to the cassette DNA sequence. Reads that aligned completely (group B and some reads [#4 and #5] in group D) are retained (green solid shaded box). Reads that are not aligned completely are discarded (green dashed box). Step 4, Reads retained from steps 2 (magenta solid box) and 3 (green solid box) are compared. Common retained reads (#4 and #5 in group D) are extracted, and the full-length reads are realigned to the Chlamydomonas reference genome. Breakpoints in these reads define the cassette insertion site (orange vertical line) in the genome.
- Figure 2.
Cosegregation of PCR-validated insertion sites, paroR, and the mutant phenotype. A, Gene structure of the Cre06.g266450 gene. Green box, 5′ Untranslated region (UTR); orange boxes, exons; black lines, introns; purple box, 3′ UTR; blue triangle, insertion site of the cassette; arrows, positions of the primers used in B. The lengths of the blue triangle and arrows are not drawn to scale. B, Insertion of a truncated cassette allows PCR amplification in both mutant and wild-type cells. In six progeny from an octad between 7F3 and CC-125, the primers flanking the Cre06.g266450 insertion sites amplify two different PCR products. In this octad, resistance (R) to paromomycin cosegregates with the larger band (Cre06.g266450+ins [insert]) and sensitivity (S) to paromomycin cosegregates with the smaller band (Cre06.g266450). C, Gene structure of the BAR1 gene. Arrows indicate the positions of the primers used in D. The lengths of the blue triangle and arrows are not drawn to scale. D, The multiciliary phenotype in 1D11 always cosegregates with the insertion in BAR1, as shown in eight random progeny. The absence of a PCR product cosegregates with multiciliary cells (M) and the presence of a PCR product is always found in biciliary cells (B). E, The 1D11 mutant assembles multiple cilia. An antibody to α-tubulin (green) stains cilia protruding outside the round cell body. Bar = 10 µm.
- Figure 3.
Complex DNA rearrangements in multiple insertional strains. A, Diagram of chimeric DNA formed across different chromosomes in five strains (yellow, 9H4; red, 3F8; purple, 8C12; green, 6A12; blue, 9A9). The ends of the arcs indicate the junction sites. Chromosomes 1 to 17 and scaffolds 18 to 54 are drawn to scale in a clockwise direction. B, Positions of the primers in the wild type and in the mutant that form chimeric DNA due to DNA duplication or DNA translocation. Expectations for events on two different chromosomes (A, green; B, magenta) are shown. We assume that the insertion happens on chromosome A and the inserted DNA originates from chromosome B. C, Expected outcome of PCR products from the wild type and mutants with different combinations of primers. D, PCR of wild-type fragments (1F-1R on chromosome 2, 2F-2R on chromosome 8) and a chimeric DNA fragment (1F-2R) in the wild type (CC-124 and CC-125), 8C12, and 9D5. Primers for the mating-type loci (MT, including the MTA1 and MTD1 genes) are included as loading controls. No temp indicates that no DNA template was added to the PCR. E, PCR of wild-type fragments (1F-1R on chromosome 1, 2F-2R on chromosome 12) and a chimeric DNA fragment (1F-2R) in the wild type (CC-124 and CC-125), 8D6, and 9H4.
- Figure 4.
Complex DNA rearrangement adjacent to the APHVIII cassette. A, Diagram of events in 3F8 involving chromosome 2 (green) and chromosome 12 (magenta) in the wild type, chimeric DNA between chromosomes 2 and 12 (#1), between chromosome 2 and the APHVIII cassette (blue; #2), and between chromosome 12 and the APHVIII cassette (#3). The positions of the primers used in B along the chromosomes and the cassette are indicated by short arrows. The orientation of the DNA fragments along the wild-type chromosome (from small to large coordinates) is indicated by long arrows. The diagram is not drawn to scale. A possible DNA rearrangement event in 3F8 is indicated at the bottom. The dashed line indicates the undetermined composition of DNA. B, PCR amplification of multiple DNA fragments in the wild type (CC-124 and CC-125), 3F8, and 3D1. No temp indicates that no DNA template was added to the PCR. C, Diagram of events in 6A12 involving chromosome 5 (green) and chromosome 6 (magenta) in the wild type, chimeric DNA between chromosomes 5 and 6 (#1), between chromosome 5 and the APHVIII cassette (#2), and between chromosome 6 and the APHVIII cassette (#3). The positions of the primers used in D along the chromosomes and the cassette are indicated by short arrows. The orientation of the DNA fragments along the wild-type chromosome (from small to large coordinates) is indicated by long arrows. The diagram is not drawn to scale. A possible DNA rearrangement event in 6A12 is indicated at the bottom. The dashed line indicates the undetermined composition of DNA. D, PCR amplification of multiple DNA fragments in the wild type (CC-124 and CC-125), 6C2, and 6A12. No temp indicates that no DNA template was added to the PCR. E, Diagram of events in 9A9 involving chromosome 9 (green) and chromosome 13 (magenta) in the wild type, chimeric DNA between chromosomes 9 and 13 (#1), between chromosome 9 and the APHVIII cassette (#2), between chromosome 13 and the APHVIII cassette (#3), and between regions of chromosome 13 (#4 and #5). The positions of the primers used in F along the chromosomes and the cassette are indicated by short arrows. The orientation of the DNA fragments along the wild-type chromosome (from small to large coordinates) is indicated by long arrows. The diagram is not drawn to scale. A possible DNA rearrangement event in 9A9 is indicated at the bottom. The dashed line indicates the undetermined composition of DNA. F, PCR amplification of multiple DNA fragments in the wild type (CC-124 and CC-125), 7F3, and 9A9. No temp indicates that no DNA template was added to the PCR.
- Figure 5.
Distribution of 49 new molecular markers for meiotic mapping. Newly designed molecular markers and their positions along different chromosomes are indicated by magenta vertical lines. Their positions along the chromosomes in version 5.5 are indicated above the line. One marker, which maps to both chromosome 12: 5.68 Mb and chromosome 16: 1.21 Mb, is indicated as light blue vertical lines. A subset of previously defined genes used in meiotic mapping is indicated by black vertical lines. Centromeres on each chromosome are indicated as green circles as mapped in Supplemental Table S5. We did not identify centromeres on chromosome 11 or 15.
Tables
Pairs of Strains Chromosome Breakpoint Range No. of Reads Insertion Gap Gene Identifier Gene Name Mutant in Strain 1 Mutant in Strain 2 bp 1A4-1D4 2 Unknown–6,876,017 1 Open ended Cre02.g119651 Cre02.g119651 Yes No 3 4,539,389–4,539,406 6 18 Cre03.g176651 MYSM1 No Yes 17 4,322,754–4,322,816 6 63 Cre17.g730950 FLA10 Yes No 2F1-4C2 1 6,737,064–6,737,071 11 8 Cre01.g048400 DZIP1L Yes No 3 7,296,662–7,296,698 11 37 Cre03.g206950 Cre03.g206950 Yes No 5 1,027,021–1,027,042 4 22 Cre05.g246553 Cre05.g246553 Yes No 17 3,105,419–3,105,428 11 10 Cre17.g721250 FAP22 No Yes 3F8-3D1 1 4,336,001–4,336,135 9 135 Cre01.g029400 TRPC3 No Yes 2 7,226,958–7,227,798 9 841 Cre02.g145950 Cre02.g145950 Yes No 2 7,290,053–unknown 2 Open ended Cre02.g145500 PTK24 Yes No 4 1,568,203–1,568,528 8 326 Cre04.g213400 SUB7 Yes No 10 4,519,930–4,520,053 4 124 No predicted gene No predicted gene Yes No 12 7,660,573–unknown 8 Open ended No predicted gene No predicted gene Yes No 17 Unknown–768,566 3 Open ended No predicted gene No predicted gene No No 17 768,925–unknown 7 Open ended No predicted gene No predicted gene No No 5E3-5F11 6 5,109,304–5,109,339 6 36 Cre06.g281050 VPS5A No Yes 9 2,129,462–2,129,485 3 24 Cre09.g393551 Cre09.g393551 Yes No 16 6,582,926–6,582,930 8 5 Cre16.g672200 BLD11 Yes No 8D6-9H4 14 1,860,469–1,860,478 14 10 Cre14.g620500 Cre14.g620500 No Yes 16 6,583,673–6,583,708 12 36 Cre16.g672200 BLD11 Yes No 16 5,635,438–5,635,438 15 0 Cre16.g679550 FAP277 No Yes 8C12-9D5 2 1,736,814–unknown 7 Open ended Cre02.g086300 Cre02.g086300 No Yes 6 5,398,524–5,398,526 13 3 Cre06.g283900 TMEM45B Yes No 9 6,286,926–6,286,995 9 70 Cre09.g406500 Cre09.g406500 No Yes 16 Unknown–6,571,444 1 Open ended Cre16.g672250 MPA13 No No 6C2-6A12 5 1,676,418–unknown 2 Open ended Cre05.g242850 Cre05.g242850 No Yes 6 Unknown–3,512,412 1 Open ended Cre06.g278107 Cre06.g278107 No Yes 6 4,339,867–4,339,917 13 51 Cre06.g278262 Cre06.g278262 Yes No 12 762,534–unknown 2 Open ended Cre12.g495500 Cre12.g495500 No Yes 6F2-6B10 16 1,141,900–unknown 2 Open ended Cre16.g650200 MITC17 Yes No 16 1,266,125–1,266,130 15 6 Cre16.g651350 ATG11 No Yes 7F3-9A9 6 2,254,537–2,254,612 19 76 Cre06.g266450 Cre06.g266450 Yes No 6 1,938,823–1,938,849 10 27 Cre06.g263650 Cre06.g263650 Yes No 9 Unknown–497,660 3 Open ended Cre09.g404201 PHC10 No Yes 13 3,741,836–unknown 3 Open ended Cre13.g589450 Cre13.g589450 No Yes 17 3,468,906–3,468,911 9 6 No predicted gene No predicted gene No Yes 1D11-6D11 11 1,722,733–1,722,733 21 0 Cre11.g467784 SOUL Yes No 16 1,549,854–1,549,855 11 2 Cre16.g653450 BAR1 Yes No 17 Unknown–5,178,817 3 Open ended Cre17.g735550 Cre17.g735550 No Yes 17 5,244,723–unknown 1 Open ended Cre17.g736000 Cre17.g736000 No Yes 17 Unknown–5,430,365 4 Open ended Cre17.g736800 Cre17.g736800 Yes No - Table 2. Experimentally verified breakpoints by meiotic mapping
n/a, Not available; ND, not determined.
No. of Inserts Strain Phenotype Chromosome Gene Name Location in Gene Cosegregation with paroRa Cosegregation with Phenotypea Linkage with paroR 1 1D4 n/a 3 MYSM1 Intron 9 5/5 n/a Yes 1 4C2 n/a 17 FAP22 3′ UTR 8/8 n/a Yes 1 5F11 n/a 6 VPS5A Exon 3 11/11 n/a Yes 1 6C2 n/a 6 Cre06.g278262 Exon 4 9/9 n/a Yes 1 6F2 Slow growth 16 MITC17 Intron 1 9/9 9/9 Yes 1 6B10 n/a 16 ATG11 3′ UTR 12/12 n/a Yes 1 8C12 n/a 6 TMEM45B 5′ UTR 16/16 n/a Yes 1 8D6 No cilia 16 BLD11 Intron 14 + exon 15 24/24 24/24 Yes 2 1A4 No cilia 17 FLA10 Exon 18 11/11 11/11 Yes 2 5E3 No cilia 16 BLD11 Exon 13 3/3 3/3 Yes 2 6D11 n/a 17 Cre17.g735550 5′ UTR 10/10 n/a Yes 17 Cre17.g736000 Exon 5 10/10 n/a Yes 2 7F3 n/a 6 Cre06.g266450 Intron 3 11/14 n/a No 6 Cre06.g263650 Exon 13 14/14 n/a Yes 2 9D5 n/a 2 Cre02.g086300 5′ UTR 9/9 n/a Yes 3 1D11 Multicilia 11 SOUL 3′ UTR 3/3 n/a Yes 16 BAR1 Intron 5 96/96b 96/96b Yes 17 Chr17-4.678M n/a 1/3 n/a No 3 2F1 Paralyzed cilia 1 DZIP1L Exon 18 12/12 12/12 Yes 3 6A12 n/a 5 Cre05.g242850 3′ UTR 10/10 n/a Yes 6 Cre06.g278107 5′ UTR 10/10 n/a Yes 3 9A9 n/a 13 Cre13.g589450 Intron 1 1/6 n/a No 17 No predicted gene n/a 6/6 n/a Yes 5 3F8 No cilia 2 Cre02.g145950 Exons 5 to 8 + introns 5 to 7 8/8 8/8 Yes 2 PTK24 3′ UTR 8/8 8/8 Yes 4 SUB7 Introns 1 and 2 + exon 2 3/8 3/8 No 12 No predicted gene n/a 8/8 8/8 Yes
Supplemental Data
Supplemental Tables and Data
Files in this Data Supplement:
- Supplemental Data - Supplemental Tables 1-6 and Supplemental Data 1-3
